New investigations on the guaiacol peroxidase of Opuntia ficus indica L. and its modulation by ascorbic acid and copper. Towards an optimization of quantitative and qualitative tests

• Peroxidases were tested in cladode extracts of 10 Opuntia ficus indica ecotypes.
• Ecotypes differed by their peroxidase kinetics, using guaiacol and o-dianisidine.
• 80% of ecotypes, showed lag time-typified peroxidase kinetics, for guaiacol.
• Ascorbic acid induced a lag time kinetic for guaiacol peroxidase.
• Copper eliminated lag time kinetics and allowed the revelation of new isoperoxidases.

When compared with o-dianisidine peroxidase, guaiacol peroxidase extracted from acetone powder prepared from cladodes of 10 ecotypes of Opuntia ficus indica L., was found to be less active in 90% of total ecotypes. In addition, lag time kinetics altered seriously guaiacol peroxidase tests. A part of 80% of ecotypes showed lag time kinetics with guaiacol substrate versus 10% for o-dianisidine. Filtered extracts on Sephadex G-25, containing low amounts of small molecules (such as antioxidants), did not show lag phases. This data support the hypothesis of involvement of small size antioxidants in the appearance of lag time. At this semi-purification level, the enzyme showed low affinity towards guaiacol. In addition, adding ascorbic acid (AsA) as exogenous antioxidant, to the guaiacol peroxidase reaction medium, led to the reappearance of lag time, where its increase follows increases in AsA concentrations. A positive correlation (coefficient 0.86) between lag times and AsA concentrations was established. The addition of copper (known as oxidant of ascorbic acid) in the reaction mixture affected the lag phase behaviour. At a final concentration of 0.5–1.0 mM, Copper abolished completely a lag time of about 120 s, allowing guaiacol peroxidase reaction to start immediately. Electrophoretic analysis of acidic peroxidases on 6–15% gradient polyacrylamide gels followed by in situ revelation using reaction mixtures containing 0.5–1.0 mM Cu++, allowed the appearance of new fast isoforms (Rf 0.66–0.68) which are absents in controls. Contrarily, Staining gels with substrate solutions containing 0.10 mM and 0.15 mM AsA, led to the disappearance of isoperoxidases at zones A1 (Rf 0.29), A2 (Rf 0.52) and A4 (Rf 0.72). A time-dependant and zone-dependant reappearances of isoperoxidases were noted after 30 min incubation. These results are discussed in respect of optimizing quantitative and qualitative aspects of guaiacol peroxidase in Opuntia ficus indica L.

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